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is the ability to create the same measure over and overwithout variation

iii)

Instruments arecalibratedso they are accurate; when you usethem correctly, your measurements are precise.

b)

Microscope

–

a microscope is an instrument that uses lenses tomagnify the image of an object

i)

Magnifying glass-

simple microscope (one lens)

ii)

Light microscope–

compound microscope (twolens)

(1)

www.udel.edu/scope

(2)

Path of light throughmicroscope

(3)

Inversion–

As aresult of light wavesbeing bent as go through the lens, theimage at the eye piece is upside

downand backwards

(4)

Resolution

-

the ability to tell two objects apart. The betterthe resolution, the clearer the picture (refer to TV news whereface of alleged criminal is pixelated to prevent identify).

(5)

Powers of magnification

–

the times larger an image is than theoriginal object. In simple microscopes, it is the power of thelens (2X is twice as large as real object). In a compoundmicroscope, there are two lenses so the total magnification isthe product of the powers of the two separate lenses.

Ocular Lens

ObjectiveLens

TotalMagnification

called

10X

4X

40X

scanning (low)Power

10X

10X

100X

medium power

10X

40X

400X

high dry power

10X

100X

1000X

High oil (must use oil)

c)

Pipet

An instrument made of a glass tube and a bulb that canmeasureor

transfer precise volumes of a liquid by drawing the liquid up intothe tube.

d)

Micropipet

–

an instrument thatcan measure or transfer preciseverysmall volumes of a liquid–

usually less than 1 millileter.

e)

Scale/balance-

An instrument can measure precise masses of a liquidor solid.

f)

Graduated cylinders

-An instrument made of a glass cylinder canmeasureprecise volumes of a liquid.

g)

Note:Beakers

andflasks

are NOT calibrated so they are NOT usedfor measuring. Observe side of beaker/flask “+/-

5%”-

this meansthe numbers on the glassware can be off as much as 5%

2)

Solutions and Media–

solutions arehomogeneous mixture

of two or moresubstances (liquid and liquid, liquid and solid, liquid and gas)

a)

Solutions

i)

Terms–

(1)

Solvent

–

liquid into which something is dissolved

(2)

Solute

–

the solid that is dissolved in the solvent (if two liquids,the one in greater amount is the solvent)

ii)

Percent

(1)

A solution that is based on the amount of solute divided by thetotal volume of the solution. In example below, solvent is water

(a)

Mass/ volume (read as mass per volume)-

5g of NaCl in1O0 mls final volume is a 5% solution. (note–

100 mls finalvolume, NOT added to 100 mls. Why?)

(b)

mass/mass–

5g of NaCl in 100g solution is a 5% solution

(c)

volume/volume–

10 ml of Ethanol in 100 mls final volume is a10% solution

b)

Molar

–

a molar solution is one mole in a final volume of 1 liter solution.

i)

a mole (6.023 X 1023

molecules

–

Avogodro’s number) is a ‘chemist’sdozen’–

if you have a mole of molecules you have 6.023 X 1023

-

justlike if you had a dozen molecules you’d have 12.

ii)

A mole is based on the number of particles

(1)

so a mole of one particle type can has a different mass thananother

(a)

Think of this: if you had a dozen pingpong balls and a dozenbowling balls, you’d have 12of each BUT if you weighedthem, the 12 bowling balls would weigh a lot more than the12 ping pong balls. So …… if you have a mole of pingpongballs and a mole of bowling balls, which would weigh more?

(b)

Now let’s look at molecules–

let’s take water (H2O) andcarbon dioxide (CO2). Do you remember how to find theformula (molecular) weight? Add up the weights of theatoms in the formula–

water is 18 (2 H at 1, one O at 16) andCarbon dioxide is 44 (1C at 12, 2 O at 16). Here’s the coolpart–

inorganic nutrients. Lions are carnivores and requiremeat. Cows are Herbivores and need grasses. People are omnivoresand eat their nutrients in both plant and animals form. Do yourememberautotrophs

andheterotrophs?

Bacteriacan be either autotrophs or heterotrophs. Autotrophicbacteria are cyan bacteria–

sometimes mistakenly called algae. Mostbacteria are heterotrophs. Some live by absorbing nutrients fromdead materials (decomposers), some from livematerials–

the bacteriathat give you zits, for instance. Some live off dead material inside you-

theE.coli

that are found in your gut–

but the living conditions are atrade–

they produce Vitamin K that you absorb.

Could you identify a virus as anautotroph or heterotroph?

When we want to grow bacteria in a laboratory–

not in their naturalenvironment, we must supply them with not only the nutrients thatneed to grow and reproduce, but also the correct environmentalconditions–

temperature, pH, salt concentrations. Since bacteria livein and on their food, the food also provides the pH and saltconcentrations. There are two basic type of food….

i)

Broth–

a nutrient mixture in liquid form.

ii)

Solid (agar)–

nutrient mixture that has base added–

usually agar–

that allows the media to solidify. Agar is usually used because it isnot a nutrient for most bacteria–

so it doesn’t change the nutrientcontent of the media. Gelatin is sometimes also used to solidifymedia, but it is a protein source for most

bacteria.

iii)

Petri Dishes are usually filled with agar media that is 1 to 1.5%agar. (So, how many grams of agar are used to make 100 mls ofagar media? Why is this not a molar solution?)

iv)

Motility agar–

is less dense (why?)–

is usually 0.5% agar.

v)

Agar is a polysaccharide–

purified from Brown Kelp-

the samestuff you see on the beaches. A similar agar is used in Japanesefoods (agar-agar) and ice cream as a thickener.

(1)

Powdered agar is yellow in color; a long stringymolecule that looks like a balled up piece ofstring

(2)

When agar is added to liquid and heated, thepolysaccharide molecules move into long strings,and then as they cool, they reform the ball, but because theyare in solution, they curl around each other. With a largenumber of molecules (1 to 1.5%) the agar becomes relativelysolid. Lower numbers of molecules means the agar is softer(0.5%).

(3)

Agarose, which we will talk about later during electrophoresis,is a highly purified form of agar. It is pure white.

d)

Solution

–

There are some

solutions that are made in small amounts–

usually because the solute is either very expensive, or not stable insolution (meaning you can’t store it so making a lot is wasteful.) Inmicrobiology and biotechnology, the two most common of these areenzymesolutions and antibiotic solutions. They are usually made inmg/ml.

i)

Enzymes are proteins that have a catalytic function–

they speedup specific chemical reactions. Examples are restriction enzymesthat cut DNA or ligase that can make the sugar-phosphate bondsand put the DNA back together. As proteins, they are made insolvents that have the optimum pH and salt concentrations. Verylittle of the enzyme solution is used when setting up reactionsbecause enzyme molecules can work over and over and over withoutdamage.

ii)

Antibiotics–

antibiotics are substances that kill bacteria.Antibiotics can be purchased in powdered or liquid form.Antibiotics are usually added directly to the agar or broth whenthe temperature is approximately 40C. If the temperaturehotter, it can damage the antibiotics so they don’t function.

3)

Growing/Counting Bacteria-

Bacteria grow fast–

they can reproduceevery 20 minutes under optimum conditions. To study bacteria or to usethem to produce products, scientists need to isolate pure bacterialstrains–

a culture that has only one type of bacteria in it, and sometimesever harder, they need to keep that strain pure as they work with it.Contamination by unwanted microorganisms is very common. So rememberthis rule when working with bacteria:

Just because it looks clean doesn’t mean it’s sterile

The corollary: What you can’t see CAN hurt you–

or at least your culture.

A few terms may help here:

Sterile

–

means without life of any kind

Disinfected

–

means to reduce or inhibit growth of organisms

a)

Sterile technique

i)

http://www.mc.uky.edu/oaa/curriculum/iid98/manual/00labtechniques.htm

ii)

Also see PDF Sterile Technique

iii)

Sterile technique, sometimes called aseptic technique, is a way tohandle materials in the laboratory that prevent any microorganismsfrom the environment from contaminating your cultures. Atechnique is amethod

of performance. It is a way to do things toensure no outside microorganisms enter your cultures. Some partsof sterile technique are easy–

like autoclaving materials andequipment to make sure they are sterile. Some parts are harder–

like transferring a pure culture from one tube or plate to another.Sterile technique is hard onlybecause there are so many details tokeep track of-

but the key is to be methodical and prepare aheadof time. Take your time and make sure your equipment and areaare ready and it will save you hours of trying to re-isolate yourculture.

b)

Transfer of cultures

–

since bacteria are alive, they will run out offood (or their metabolism will create waste products and foul theirenvironment), so to keep your cultures healthy, they must beperiodically moved to new media. This usually involves transferring to

like media (liquid to liquid, slant to slant, plate to plate) using steriletechnique.

c)

Liquid cultures–

liquid cultures are used to grow many bacteria at atime. As the number of bacteria in liquid media increase, the liquidwill become cloudy.

d)

Plates for colonies

–

if you want to see the structure of a colony ofbacteria, or you want to start a liquid culture with a group of bacteriayou know are all the same, you will plate out bacteria on agar plates.Here the media is solid, and the bacteria siton top. In a spreadingtechnique, you pull bacteria across the plate using an inoculating loop,until individual bacteria are rubbed off the loop. By incubating theseplates, the bacteria start to grow. Since the media is solid, thebacteria cannot float away from each other as they do in liquid; ratherthey pile up. When there are about a million bacteria, this pile getslarge enough to see with the naked eye and we call this a colony. Eachcolony started from a single bacterium, so each colony is a clone

–

genetically identical can be used to start a pure culture. (Note: It willbe a bit different for viruses, but we’ll talk about that when we do it)

e)

Spreading Plates–

If you have liquid cultures and want to growbacteria across the entire surface of the plate (called a lawn), thenthe drop of bacteria are placed in the center of a plate and asterilized glass spreader (“hockey stick”) is used to spread thesolution across the entire plate. In this technique, there are so manybacteria next to each other

that you cannot see the individualcolonies, so it is called a lawn–

just like if you look at a lawn, you donot see the individual grass plants.

f)

Counting Bacteria usingDilutions

–

There are times you need to knowhow many bacteria you have in a liquidculture. Since there may be 109

bacteria per milliliter-

1,000,000,000–

it may be too hard to countthem directly under a microscope…. So an easy way to count them is todilute the original culture; count the bacteria in the dilution, and usesome basicmath to calculate how many were in the original culture.There are two basic ways to do this-

plate out a sample of thedilution by spreading on plates and waiting for colonies to form andcount the colonies, or count the diluted bacteria directly using a

hemocytometer

g)

Makingserial dilutions

i)

http://abacus.bates.edu/~ganderso/biology/resources/dilutions.html

ii)

To be able to count colonies on a plate, there should be(statistically) between 30 and 300 colonies, fewer than 30 and arandomness is not assured–

greater than 300 (250 is actuallybetter), it is difficult to count the colonies accurately

iii)

A dilution is where a small sample of bacteria is placed in a steriletest tube, mixed with a liquid (usually nutrient broth or saline) sothe bacteria are evenly distributed throughout the tube, and thena sample is used for counting.

iv)

Because they are SO many bacteria in the original sample, veryoften we need to dilute the dilutions to get a number of bacteriawe can count on a plate. This dilution series is called serialdilutions.

4)

Activities

a)

Using a Microscope (oil lens)

i)

http://www.udel.edu/scope

ii)

Any basic microscope lab PLUS how to use oil lens

iii)

Emphasize to use ONLY lens paper to clean lens–

regular tissuesor Q-tips can scratch lenses (scratch the coating on the lens)

iv)

Be sure to teach how to clean immersion oil off lens and need to doso to prevent damage since old immersion oil will crystallize on lenscausing damage.Use naphtha, xylene, or turpentine (use very smallamounts on lens tissue)

as solvent.

Do not use water, alcohol oracetone, as the oil is insoluble to these solvents.